1. Field of the Invention
The present invention relates generally to the fields of molecular biology and gene transfer and particularly concerns recombinant adeno-associated virus (AAV). The invention provides novel methods and compositions, including cell lines, recombinant AAV and adenovirus or herpes virus vectors, for use in the efficient and large-scale production of adeno-associated virus. The AAV production methods described herein do not require a transfection step. The resultant AAV may be used in a variety of embodiments including, for example, for transferring exogenous genes into human cell lines and for use in human gene therapy regimens.
2. Description of the Related Art
There are currently more than 4,000 known genetic disorders which lack fully effective therapies. In recent years the prospect of using gene therapy to treat such diseases has become to be viewed as a realistic goal. The ultimate form of gene therapy requires the integration of a wild-type gene able to correct the genetic disorder into the host genome, where it can co-exist and replicate with the host DNA. The expression of the gene should be regulated at a level that can best compensate for the defective gene. In the most ideal circumstances, the disease would be cured for life by one or a few treatments, with no serious side effects.
There have been several experimental approaches to gene therapy proposed to date, but each suffer from their particular drawbacks (Mulligan, 1993). Firstly, there are basic transfection methods in which DNA containing the gene of interest is introduced into cells non-biologically, for example, by permeabilizing the cell membrane physically or chemically. This approach is limited to cells that can be temporarily removed from the body and can tolerate the cytotoxicity of the treatment, i.e. lymphocytes. Furthermore, the efficiency of gene integration is very low, on the order of one integration event per 1,000 to 100,000 cells, and expression of transfected genes is often limited to days in proliferating cells or weeks in non proliferating cells.
The retroviral vector approach capitalizes on the natural ability of viruses to enter cells, bringing their own genetic material with them. Retroviruses have the advantage that they can integrate into host genome and thus transfer the gene of interest into the genome of the target cell. However, major problems are associated with using retroviral vectors for gene therapy, for example, they only integrate efficiently into replicating cells and they are difficult to concentrate and purify.
Several DNA viruses, such as adenovirus, have also been engineered to serve as vectors for gene transfer. But many DNA viruses that can accept foreign genetic material are limited in the number of nucleotides they can accommodate and in the range of cells they infect. Moreover, adenoviruses do not integrate their genetic material into the host genome and, due to the resultant transient expression, repeated exposures would be necessary. Unfortunately, the limited serotypes of adenovirus available for vector development and host immunity pose limitations on repetitive administration.
Another limitation on adenovirus vectors is the fact that recipient cells generally express at least a low level of viral proteins in addition to the therapeutic gene (Gregory et al., 1992; Rosenfeld et al., 1992), attracting immune responses and causing inflammation in the recipient organ. The co-transfer of viral genes into recipient cells also opens the possibility that the defective viral genome can be rescued by a wild-type virus infection which may propagate the genetically engineered virus and gene among the normal population. Furthermore, studies have shown that recombinant adenovirus which are meant to be replication-defective can in fact replicate slowly (Shenk et al., 1980), raising general safety concerns.
The properties of Adenoassociated Virus (AAV), a single-stranded DNA parvovirus endogenous to the human population, make it one of the most suitable gene therapy vector candidates. Firstly, AAV is not associated with any disease (Ostrove et al., amp 1981; Cukor et al., 1984), therefore it is safe for gene transfer applications. Secondly, AAV virions are resistant to physical treatments, such as sonication and heat inactivation, that are not tolerated by other viruses during purification (Samulski et al., 1989). Thirdly, like retroviruses, AAV integrates into the host cell genome upon infection (Kotin et al., 1990; Samulski et al., 1991) so that transgenes can be expressed indefinitely. Furthermore, integration of AAV into the cellular genome is independent of cell replication (Lebkowski et al., 1988). This is particularly important as AAV can thus transfer genes into quiescent cellsxe2x80x94which make up the vast majority of cells in the human body.
However, AAV technology does have certain limitations which remain to be overcome. For example, benefits may be realized by developing strategies to accommodate larger recombinant inserts. The major problem limiting the practical use of recombinant AAV is that AAV production methods are inefficient and laborious (Lebkowski et al., 1988; Samulski et al., 1989; Muzyczka, 1992). In recombinant AAV, key viral genes (such as cap, lip and rep) are replaced by the exogenous gene of interest. Methods for producing recombinant AAV therefore rely on co-transfecting the AAV vector carrying the gene of interest, together with a helper AAV plasmid that expresses all of the essential AAV genes, into adenovirus- or herpes-infected cells which supply the helper functions necessary for AAV replication and the production of new viral particles.
The use of cells infected with helper adenovirus or herpes virus does not create a problem, it is the transfection of the essential AAV genes which is the limiting step for the production of high titre AAV virus. Transfection of DNA molecules into cells is known to be very inefficient and the transfection-based methods generally used for AAV production are, therefore, particularly inefficient as they rely on co-transfection. Even the most-recent studies in this area have only reported a 10% increase in efficiency (Page et al., 1993). Unfortunately, new procedures, such as those utilizing chimeric Epstein Barr/AAV plasmids (Lebkowsi et al., U.S. Pat. No. 5,173,414) and transduced cells with AAV vectors stably incorporated into the genome (Muzyczka et al., U.S. Pat. No. 5,139,941), still require transfection of a helper plasmid that provides AAV genes for packaging. The production of a significant quantity of AAV virions for various applications, including clinical uses, using the current methodology thus remains impractical and a new procedure for the efficient production of large quantities of recombinant AAV vectors would clearly be highly beneficial.
The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing novel compositions and methods for use in the efficient, large-scale production of recombinant adeno-associated virus (AAV). The compositions of the invention include recombinant AAV vectors, AAV-producing cell lines including such vectors, and recombinant infective virus vectors capable of expressing essential AAV genes required for AAV virion assembly and genome packaging. The invention also provides advantageous methods for AAV production which utilize virus infection and do not require DNA transfection.
The following terms and definitions employed herein to refer to recombinant adenoviruses and AAV inserts follow the conventional type of nomenclature used in the art. AdAAV is used as a general term to refer to adenovirus that contains one or more AAV genes inserted in any region of the adenovirus genome, this includes replication defective and replication competent adenovirus. Adenovirus carrying a particular gene can be termed as a prefix, Ad-, when the letters representing the inserted DNA will follow the prefix. The location in the adenovirus where the exogenous DNA is inserted will be a suffix, such as -E1 or -E4. For example, AdAVlacE1 is recombinant adenovirus carrying an AAVlacZ DNA inserted in the E1 region.
The AAV rep-lip-cap gene is abbreviated as rc, the rep-lip gene is abbreviated as rep, and the cap gene is represented as cap. Therefore, AdcapE3 is the cap gene of AAV inserted into the E3 region of an adenovirus. The ITR sequences are the only essential cis-acting elements for an AAV vector to mediate genome packaging and integration into host cells. The presence of ITRs in a DNA fragment will be indicated by xe2x80x9cAVxe2x80x9d. For example, AVlac is a DNA fragment contains two ITR sequences flanking a lacZ gene. Such a DNA fragment is also referred as an AAV vector.
In first embodiments, the invention concerns novel virus constructs including essential AAV genes and recombinant infective virus particles or virions including such DNA constructs. The recombinant virus containing the novel constructs will be virus capable of infecting mammalian cells, with preferred examples being vectors and virions of the adenovirus or herpes virus families. Recombinant viral constructs of the invention will generally be adenoviral or herpes virus vectors capable of expressing essential AAV proteins, i.e., constructs containing a recombinant insert or inserts which include one or more expression regions encoding one or more essential AAV proteins. Such recombinant inserts capable of expressing essential AAV protein(s) may also be termed xe2x80x9ctranscription unitsxe2x80x9d.
Adenovirus used in this invention may be from any of the 42 different known serotypes or subgroups A-F of adenovirus, with Adenovirus type 5 of subgroup C being preferred as this is the most commonly used in the art. In addition, other viruses which are capable of serving as helper viruses for AAV replication and which are capable of receiving an AAV insert may be employed. Viruses belonging to the herpes family or class of viruses are particularly contemplated as they naturally provide AAV helper functions and can be readily engineered to express essential AAV proteins. Therefore, the term xe2x80x9cherpes virusxe2x80x9d is used in this context to particularly refer to herpes simplex virus (HSV), Epstein-Barr Virus (EBV), cytomegalovirus (CMV) and pseudorabies virus (PRV).
As used herein, the terms xe2x80x9cessential AAV genesxe2x80x9d and xe2x80x9cessential AAV proteinxe2x80x9d are intended to refer to those genes, and their encoded proteins, which are normally encoded by wild type AAV and are required for AAV replication, genome packaging and virion assembly. Naturally, when intended for use in AAV production, the adenoviral or herpes virus vector will be constructed so the inserted AAV genes complement any essential AAV genes which have been deleted from a recombinant AAV vector to allow an exogenous gene, such as a therapeutically important gene, to be inserted into the AAV vector.
Generally speaking, the adenovirus and herpes virus vectors and recombinant virions will therefore incorporate inserts with expression regions which comprise one or more of the AAV rep, lip and cap genes, and in certain preferred embodiments, will include all three of these genes. The recombinant inserts may also include other AAV sequences, but those vectors which include essential AAV genes and which lack other AAV sequences, such as, e.g., full length AAV inverted terminal repeat (ITR) sequences, will be most preferred. Infective virions expressing one or more essential AAV genes may be employed to supply these genes, and consequently, the rep, lip and/or cap proteins, to cells containing a recombinant AAV vector in which the particular essential gene or genes have been replaced by a chosen DNA segment or transgene.
Essential AAV genes may be introduced into recombinant adenovirus or herpes virus in accordance with the invention simply by inserting or adding the AAV coding sequences into the viral genome. However, more generally, an adenovirus or herpes virus gene will be deleted and the essential AAV coding sequence(s) will be introduced in its place. Any genes, whether essential for replication, such as adenovirus regions E1, E2 and E4, or non-essential for replication, such as adenoviral E3, may be deleted and replaced in this manner. Where the deleted gene is essential for replication, the resultant recombinant virus will be a replication-deficient adenovirus or herpes virus vector capable of expressing an essential AAV protein. Techniques for preparing replication-defective infective viruses are well known in the art, as exemplified by Ghosh-Choudhury and Graham (1987); McGrory et al. (1988); and Gluzman et al. (1982), each incorporated herein by reference.
Replication-defective viral vectors in accordance with the present invention will generally be constructed by deleting or removing a gene required for adenovirus or herpes virus replication and introducing in its place the desired AAV genetic material. Where this approach is taken, any gene required for adenovirus or herpes virus replication may be deleted and replaced by an essential AAV gene or genes. Suitable examples include, for instance, replacement of the adenovirus E1, E2 or E4 genes; insertion into the HSV tk gene; insertion into the PRV tk, gIII or gX glycoprotein genes; and insertion into CMV xcex1- or xcex22.7 promoter regions. Replacement of the adenovirus E1 gene is preferred in certain embodiments as this procedure is most routinely practiced in the art, however, replacement of the adenovirus E4 gene is preferred in certain other embodiments as this is contemplated to allow for particularly high titre AAV production when combined with E4 expressing cells.
The adenoviral or herpes virus vectors of the invention may direct the expression of AAV gene or genes by using either xe2x80x98natural promotersxe2x80x99, i.e., adenovirus or herpes virus promoters, or xe2x80x98heterologous promotersxe2x80x99, i.e., promoters from other sources. The choice of promoter is not believed to be particularly critical so long as the promoter effectively directs the expression of the AAV gene or genes. One may also use a constitutive promoter to ensure a high, constant level of expression of the AAV genes.
The promoter used may be from any viral or eukaryotic source, including plant and animal promoters, such as those in the following exemplary list. Promoters derived from plant and animal genes may include promoter sequences from significantly expressed genes, such as the xcex1-tubulin gene and xcex2-actin genes in plants and, by way of example, the immunoglobulin and hormone genes in animals.
Preferred promoters for use in accordance herewith include viral promoters, such as the SV40 late promoter from simian virus 40, the Baculovirus polyhedron enhancer/promoter element, Herpes Simplex Virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including LTR elements. For simplicity, the use of AAV promoters, such as the AAV P5 promoter, will generally be preferred in most cases. The vectors of the invention will also generally include a polyadenylation signal. Any suitable signal may be employed, but AAV sequences such as the AAV common polyadenylation signal are preferred.
In certain embodiments, particularly preferred vectors are replication-defective adenoviral or herpes virus vectors which include the AAV rep, lip and cap genes, along with the AAV P5 promoter and the AAV common polyadenylation signal. In other preferred embodiments, the invention provides replication-competent adenovirus vectors in which the rep-lip gene, the cap gene, or the entire rep-lip-cap unit is inserted in place of the E3 region (Adrep3, Adcap 3 and Adrc3, respectively).
In distinct embodiments, the present invention also encompasses novel virus constructs including recombinant AAV vectors and recombinant infective virions including such DNA constructs. These type of recombinant viral constructs and virions will generally be adenoviral or herpes (HSV, PRV and CMV) virus vectors and virions which have a DNA insert including an AAV vector which itself contains a recombinant insert encoding a chosen DNA segment, such as an exogenous gene or xe2x80x98transgenexe2x80x99. This is exemplified herein by the construction of pXAVlac containing the well-known marker gene lac-Z. However, any DNA segment or gene may be employed as a transgene in this regard, providing the length of the DNA segment does not significantly exceed about 5 kb in length. As with the first described vectors and viruses, ones bearing AAV vectors may also be either replication-defective, such as pXAVlac, or replication-competent, such as those in which the AAV vector is inserted into adenovirus E3, e.g., those based upon the plasmid pFGdX1.
The terms xe2x80x9ctransgenexe2x80x9d and xe2x80x9ctransgenicxe2x80x9d most often refer to an exogenous gene which has been introduced, generally, by the hand of man, into a host cell or host animal. In this sense, therefore, an exogenous DNA segment is not strictly xe2x80x98a transgenexe2x80x99 whilst it still resides within the AAV vector, rather it may properly be referred to as a recombinant DNA segment or recombinant gene. However, as AAV virions including such recombinant DNA segments are intended for use in connection with host cells and, ultimately, with host animals, the recombinant genes will later become xe2x80x98transgenesxe2x80x99 when introduced into such cells. In this sense, for simplicity, AAV vectors with recombinant DNA inserts may be termed xe2x80x9ctransgenicxe2x80x9d AAV vectors.
Recombinant, transgenic viruses and virions in accordance with this aspect of the invention may be employed to infect cells as a means of introducing recombinant AAV vectors into the cells, and preferably, to provide a stable copy of the AAV-transgene sequence which may be subsequently rescued when supplied with essential AAV functions, such as rep, lip and/or cap. The stable AAV-transgene copies may be integrated into the host cell genome or may be maintained as an episome.
In preferred embodiments, the AAV vectors described above will be non-replicative, recombinant or transgenic AAV vectors. A xe2x80x9cnon-replicative, recombinant or transgenic AAV vectorxe2x80x9d is an AAV vector which includes a recombinant insert encoding a desired protein, which vector is capable of integrating into a host cell genome but is incapable of directing its own replication and viral packaging. Such vectors will generally comprise AAV inverted terminal repeat (ITR) sequences along with an expression region encoding a recombinant protein. These vectors may also be functionally described as AAV constructs which are capable of replication when complemented in trans by the appropriate essential AAV genes.
Further aspects of the present invention concern a variety of recombinant host cells. As used herein, the terms xe2x80x9crecombinantxe2x80x9d or xe2x80x9cengineeredxe2x80x9d cells are intended to refer to cells into which a recombinant or exogenous DNA segment or gene has been introduced through the hand of man, such cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced exogenous DNA segment or gene. The recombinant host cells of the present invention will be cells which incorporate adenoviral or herpes virus vectors capable of expressing essential AAV proteins. In preferred embodiments, the vector will be introduced into the host cell by infection with recombinant virus.
Virtually any cell is considered to be suitable for use as a host cell in accordance with the present invention, with cells which are readily susceptible to infection by adenovirus or herpes virus being particularly preferred. Host cell lines are generally selected for known technical criteria such as ease of growth without complex media requirements and maintenance of inserted DNA. Useful cell lines are contemplated to include, for example, HeLa cells, KB cells, JW-2 cells, Detroit 6 cells, COS cells, CV-1 cells, VERO cells, NIH-3T3 cells and the like. Such cells are commonly available, e.g., commercially available through the American Type Culture Collection (ATCC). One example of a particularly suitable host cell which is routinely used in the art is a 293 cell (transformed primary human embryonal kidney cells).
More preferred recombinant host cells will be those which further include a stable recombinant AAV vector containing a gene of interest. xe2x80x9cStablexe2x80x9d in this context means that the AAV vector sequences reside and are maintained in a host cell, such as by being integrated into the genome, i.e., integrated into a host cell chromosome, or maintained as an episome. In certain embodiments, the most preferred cells will be those cells in which a non-replicative, recombinant AAV vector including ITR sequences and a gene or DNA segment of interest, is integrated into the genome of the cell.
The generation of recombinant AAV vectors themselves is well known to those of skill in the art. In general, the desired DNA may be ligated into the AAV genome in place of or in addition to the cap, lip or rep genes or in place of or in addition to any AAV DNA sequence excluding the first and last 145 base pairs, as described in U.S. Pat. No. 5,139,941, incorporated herein by reference. Although U.S. Pat. No. 5,139,941 describes inefficient methods for initial AAV generation, i.e., those relying on transfection, it does disclose how to make recombinant AAV vectors comprising foreign DNA suitably ligated into the AAV genome. In preferred embodiments, it is contemplated that the AAV vectors most suitable for use in accordance herewith will have exogenous DNA inserted in place of one or more of the rep, lip and cap genes, and most preferably, in place of all three such genes. Of course, the present invention is not limited to any specific exogenous gene or DNA segment, and it is contemplated that virtually any gene that one desires to insert would be suitable, so long as it is not prohibitively long, i.e., significantly longer than about 5 kb.
Recombinant host cells which include recombinant AAV vectors capable of rescue, i.e., having the potential to produce recombinant AAV virions, are termed xe2x80x9cAAV producer cellsxe2x80x9d. Preferably, producer cells of the present invention will include an AAV vector which is integrated into the genome and is capable of expressing a desired transgene. Rescue of the AAV vector will then result in the generation of recombinant AAV virions carrying the transgene, which may be employed to deliver the gene to other cells, e.g., as in gene therapy.
The xe2x80x9crescue processxe2x80x9d is the process by which the producer cells are rendered capable of producing recombinant AAV virions. This is generally achieved by supplying the cell with the AAV functions needed for replication and packaging. In accordance with the preferred methodological aspects of the present invention, rescue is achieved by infecting the producer cell with recombinant adenovirus or herpes virus encoding one or more of the essential AAV functions rep, lip and cap, as required.
Accordingly, the most preferred AAV producer cells will be those comprising a stably integrated recombinant AAV vector which includes AAV ITR sequences and an expression region encoding an exogenous gene, the cell being capable of producing recombinant AAV virions when contacted with replication-deficient adenovirus or herpes virus particles which include a vector capable of expressing one or more AAV proteins essential for AAV replication, genome packaging and virion assembly.
As will be discussed in detail herein, it should be noted that the present invention encompasses methods for AAV production which do not, technically, require the prior production of a xe2x80x9cproducer cellxe2x80x9d in the sense that this term is most commonly used in the art. These are the processes by which a host cell is simultaneously contacted with the AAV vector and the required AAV essential genes, including simultaneous transfection/infection processes and, also, double and triple infection procedures. However, the execution of such methods still, of course, results in the generation of a cell which includes a recombinant AAV vector and which is capable of producing recombinant AAV.
Further and particularly important aspects of the present invention concern novel methods for producing recombinant AAV virions, which methods are especially advantageous as they are not limited by transfection. To produce AAV virions according to the methods of the present invention, one would generally introduce into a host cell a recombinant AAV vector, and infect the cell with recombinant adenovirus or herpes virus capable of expressing one or more essential AAV proteins, i.e., those required for replication, packaging and assembly. Then one would culture the cell under conditions and for a period of time effective to allow the cell to produce recombinant AAV virions. The process of xe2x80x9cintroducing into a host cell a recombinant AAV vectorxe2x80x9d may be the process by which a producer cell is formally created, as described above, or it may be a simultaneous transfection/infection or double or triple infection process, as described below.
In preferred embodiments, the AAV production methods of the invention involve, first, preparing a recombinant adenovirus or herpes virus which includes a vector construct capable of expressing an essential AAV protein and, second, preparing a cell capable of producing AAV by introducing a recombinant AAV vector into a host cell. The AAV vector will preferably be a non-replicative, recombinant AAV vector including AAV ITR sequences and a transgene of interest which is capable of being stably maintained by the cell. One would infect this cell with the recombinant virus in an amount effective to stimulate the production of recombinant AAV virions, and culture the infected cell to obtain the virions so produced.
The preparation and purification of recombinant AAV virus is described in U.S. Pat. No. 5,173,414, incorporated herein by reference. Although the methods for initial AAV production described in U.S. Pat. No. 5,173,414 rely on transfection, and are therefore inefficient, this reference teaches how to prepare AAV, i.e., how to xe2x80x9charvestxe2x80x9d the virus from cells capable of producing AAV. Further suitable methods for harvesting the virus will be known to those of skill in the art in light of the present disclosure and articles such as, for example, Samulski et al. (1987). Adenovirus may be removed by any suitable techniques, including heat inactivation, CsCl gradient sedimentation or chromatographic techniques.
The cell capable of producing AAV may be supplied with essential AAV proteins by infection with any one of, or a combination of, a variety of recombinant infectious viruses. For example, a single virus which may employed which includes a vector construct expressing the AAV rep, lip and cap genes. Alternatively, one recombinant virus which includes a vector expressing the AAV rep-lip genes may be used in conjunction with a second recombinant virus including a vector expressing the AAV cap gene. In either case, the AAV genes may be introduced at any point of the adenovirus or herpes virus.
The recombinant infective virus used in accordance herewith may be adenovirus, HSV, PRV, CMV and the like, and may be either a replication competent virus or a replication-defective virus, such as a replication-defective adenovirus. Such viruses will generally be replication-defective as a result of deleting essential genes in order to create space for an essential AAV gene or genes be inserted. Where replication defective viruses are employed, the defect may be complemented by using an AAV-producing cell which directly expresses the protein or proteins required. Alternatively, the AAV-producing cell may be infected with a second, or even a second and third recombinant virus, which complements the defect.
By way of example, one may replace the E1 region of an adenoviral construct with a recombinant insert including the AAV rep, lip and cap genes or any one of such genes. To complement this virus, one may use a producing cell which expresses E1, such as an E1-expressing 293 cell, or one may co-infect a cell with a second adenovirus which includes a functional E1 region. The second adenovirus may be of any type so long as it directs the expression of E1 proteins. It may be one in which other functional units, including essential AAV genes or selected transgenes, have been introduced into regions such as the E3 or E4 regions.
The combination of viruses and AAV producing cells is, of course, not limited to E1-lacking virus and E1-expressing cells. Indeed, any complementary combination of viruses and host cells may be employed in connection with the present invention. For example, where the recombinant virus lacks functional E2, an E2-expressing cell or second virus may be used, where the recombinant virus lacks functional E4, an E4-expressing cell or second virus will be effective, and the like. Where a gene which is not essential for replication is deleted and replaced, such as, for example, the E3 gene, this defect will not need to be specifically complemented by the host cell.
In complementing the replicative defect of an infective virus by co-infection with another virus, the use of E3-lacking recombinant adenovirus is particularly preferred. For example, recombinant adenovirus which bear AAV genes in place of the E3region may be used in combination with recombinant adenovirus that carry an AAV transgenic vector in place of the E1 gene. This allows high levels of AAV to be produced without need of special cell lines and is advantageous in that it renders the lower level of E1 protein expression in cell lines, such 293 cells, of little consequence.
The present invention encompasses four broad methods for preparing AAV virions based upon the virus and AAV-containing cell compositions described above. In all these methods the most preferred AAV vector will be a non-replicative, recombinant AAV vector including AAV ITR sequences and a gene of interest which is capable of integrating into the host cell genome on introduction. The methods encompassed by the invention are variations based upon the method of initially introducing an AAV vector into a cell in order to create a cell capable of producing AAV.
In a first example, a typical AAV producer cell is prepared by transfecting a host cell with the chosen recombinant AAV vector, this may be referred to as xe2x80x9cprior introductionxe2x80x9d. This may be achieved by any suitable physical or chemical method, such as, for example, Ca2+-, liposome-or protein conjugate-mediated transfection, via electroporation, or indeed, via any other known method. In a second example, the cell capable of producing AAV is formed by transfecting a cell with an AAV vector and, at the same time, infecting the cell with recombinant adenovirus or herpes virus expressing essential AAV proteins, this may be referred to as xe2x80x9csimultaneous introductionxe2x80x9d. In this method, the infectious virus will generally also promote the efficient transfer of the AAV vector, particularly as it helps vector DNA escape from lysosomes.
In a third example, termed xe2x80x9cdouble infectionxe2x80x9d, a cell is simultaneously infected with two forms of recombinant adenovirus or herpes virus, one of which will express essential AAV proteins, and the other which will include a non-replicative, recombinant AAV vector encoding a desired protein and capable of integrating into the genome. A further variation of this is the fourth example which may be termed the xe2x80x9ctriple infectionxe2x80x9d method. In this method, cells-are infected with three forms of recombinant adenovirus or herpes virus, the combination of which results in the expression of all the essential AAV proteins and also provides the non-replicative, AAV vector encoding the desired protein.
In double and triple infection methods, the most preferred combinations are those in which the AAV genes and the AAV vector are inserted at different regions of the recombinant adenoviruses. This is exemplified by using AdAVlacE1 with AdrcE4, AdAVlacE1 with Adrc3 or AdAVlacE1 with AdrepE3/AdcapE3. In these cases, when the adenoviruses co-infect the host cells, their defects are complemented by each other. Also, as AdrepE3, AdcapE3 or Adrc3 will direct the production of E1, the above combinations of constructs may be used in methods to produce recombinant AAV using any cells that are susceptible to adenovirus infection, such as BK or HeLa cells, and are not limited to E1-expressing cell lines.
Further aspects of the present invention concern recombinant AAV virions and virus stocks prepared by any of the methods described herein and pharmaceutical compositions comprising such recombinant AAV virions in pharmacologically-acceptable formulations. The pharmacologically-acceptable vehicles may be buffered saline solutions rendered essentially free of undesirable contaminants, such as adenovirus particles or endotoxins and other components which may cause untoward reactions in a recipient animal or individual.
The recombinant AAV vectors of the invention and the virions produced by methods in accordance with the invention may include any desired DNA segment or gene, with the general limitation that it be of about 5 kb in length or less, or preferably about 4.7 kb in length or less, to allow effective packaging into virus particles. Apart from the size considerations, it will be understood that the invention does not limit in any way the choice of the exogenous DNA segment or gene. DNA from any source may be employed, and both coding and anti-sense constructs may be used. The recombinant AAV may be employed in any one of a variety of embodiments, such as, e.g., in introducing a desired gene into a cell in vitro, or used as a delivery vehicle for gene therapy in vivo wherein sense, or antisense, genetic constructs are delivered to cells within an animal, such a human subject.
In still further embodiments, the invention concerns AAV vector constructs comprising a full length cystic fibrosis transmembrane conductance regulator (CFTR) gene which are capable of expressing the entire CFTR protein, recombinant AAV virions including such CFTR constructs, pharmacologically-acceptable formulations of such AAV virions, and methods for their preparation and use. Although the invention is clearly not limited to aspects connected with the CFTR gene, it does, in further exemplary embodiments, provide methods for treating cystic fibrosis. Such methods generally comprise administering to a mammal with symptoms of cystic fibrosis an effective amount of a pharmacologically-acceptable composition comprising recombinant AAV virions which include a recombinant AAV vector construct capable of expressing the entire CFTR protein. The animals treated in this manner may generally be any mammal suspected of having mutations within the CFTR gene, including, of course, human subjects.